Fetal alcohol spectrum disorders: experimental treatments and strategies for intervention.

Despite the known damaging effects of prenatal alcohol exposure, women continue to drink during pregnancy, creating a need for effective interventions and treatments for fetal alcohol spectrum disorders (FASD). Experimental models can be useful in identifying potential treatments, and this article describes the spectrum of experimental therapeutics that currently are being investigated, including pharmacological, nutritional, and environmental/behavioral interventions. Some treatments target the underlying mechanisms that contribute to alcohol-induced damage, protecting against alcohol's teratogenic effects, whereas other treatments may enhance central nervous system plasticity either during alcohol exposure or long after alcohol exposure has ceased. The insights gained to date from experimental models offer several candidates for attenuating the deficits associated with FASD.

A lcohol consumption during pregnancy is the most common preventable cause of birth defects in the Western world, producing a range of physical, neurological, and behavioral alterations known as fetal alcohol spectrum disorders (FASD). Although warning labels and other public health messages have been issued, some pregnant women and women of childbearing age continue to drink (Centers for Disease Control and Prevention 2009), making it necessary to seek out treatments that might mini mize or attenuate FASD. Identifying effective interventions is among the top priorities of FASD research.
An increasing number of clinical studies are examining potential behavioral and educational interven tions for FASD (see the article by Paley and O'Connor in this issue,. Basic science models also have been useful in identifying treatments that may be translated to clinical practice. When investigating potential treatments, researchers can choose sev eral strategies. One strategy is to identify the numerous mechanisms that con tribute to alcohol's teratogenic effects and to block or prevent those actions. A second approach is to identify pro tective and provocative factors that may be controlled to improve outcomes. For example, maternal alcoholism may lead to malnutrition, which can exacerbate alcohol's damaging effects, suggesting that nutritional interventions might reduce FASD. Another approach is to use what is known of factors that have beneficial effects on development and/or to enhance neuronal plasticity, even after the alcoholinduced damage is complete. This article will summarize the latest experimental therapeutics cur rently being investigated. Although identified primarily in animal models of developmental alcohol exposure, these treatments show promise in atten uating some FASD. because it leads to higher blood alcohol concentrations and is linked to increased episodes of alcohol withdrawal. Alcohol interacts with a number of neuronal membrane binding molecules (i.e., receptors), including the NmethylD aspartate (NMDA) receptor, which normally is activated by the excitatory brain chemical (i.e., neurotransmitter) glutamate. The NMDA receptor plays an important role in neuronal plastic ity during development and later in life during learning. However, if it becomes overactive it can lead to increases in intracellular calcium and consequent cell death, a process called excitotoxicity. Alcohol acutely blocks the NMDA receptor, but chronic exposure may lead to a compensatory increase in either receptor number or glutamate release, actions that may contribute to tolerance (Crews et al. 1996;Grant and Lovinger 1995;Lovinger 1993). However, when alcohol is eliminated during withdrawal peri ods, it is postulated that overactivity of the NMDA receptor leads to exci totoxic cell death. (Tsai and Coyle 1998). This process also may occur in the developing brain (see figure 1).
Consistent with this mechanism, administration of NMDA receptor antagonists, drugs that block the NMDA receptor (e.g., MK801,agmatine,eliprodil,memantine) during the withdrawal period, can attenuate some of alcohol's teratogenic effects. For example, Thomas and colleagues (2001) demonstrated that MK801 administration during alcohol withdrawal in developing rats reduces the severity of alcoholrelated hyper activity observed in an open field, improves the animals' ability to react to changing contingencies (i.e., reversal learning), and protects against alcohol induced hippocampal cell loss, but only when administered during the withdrawal phase and at low doses (Thomas et al. 2001) (see figure 2A).
When administered at the same time as alcohol, MK801 is highly toxic and exacerbates alcohol's effects (Thomas et al. 2001). Fortunately, there are other NMDA receptor antagonists that may be safer than the potent MK801. Drugs that act on the polyamine modulatory site of the NMDA receptor (e.g., eliprodil, agmatine) have attenuated deficits on a spatial discrimination reversal learn ing task (Thomas et al. 2004a) (see figure 2B) and motor balance deficits associated with early neonatal alcohol exposure (Lewis et al. 2007). More recently, Idrus and colleagues (2011) demonstrated that memantine, a drug believed to block NMDA receptor overactivation without blocking normal neurotransmission, mitigates alcoholrelated motor coordination deficits, with concomitant neuropro tection against alcoholinduced cerebel lar neuronal loss. Similarly, in vitro studies indicate that memantine is Figure 1 NmethylDaspartate receptor (NMDAR)mediated excitotoxic cell death. During alcohol withdrawal, it is postulated that overactivity of the NMDAR leads to excitotoxic cell death in the developing brain. A) Alcohol directly interacts with the NMDA receptor, which is activated by glutamate. Acute alcohol exposure inhibits the receptor, which contributes to the sedative and intoxicating effects of alcohol. B) Continued alcohol exposure may produce an adaptive neurocompensatory response, either as an increase in the number of NMDARs or an increase in the amount of glutamate released, which contributes to acute tolerance to alcohol's intoxicating effects. However, when alcohol is elim inated from the body during periods of withdrawal, there may be rebound overactivity of NMDARs (C), which may lead to cell death (D).
neuroprotective against hippocampal pathology associated with prenatal cell loss associated with developmental alcohol exposure. alcohol exposure (Stepanyan et al. 2008). These findings suggest that Serotonin Agonists blocking NMDA receptors during withdrawal in the fetus or newborn Alcohol also acts on other neuro may protect against some of the neuro transmitter systems, including the Figure 2 NmethylDaspartate receptor (NMDAR) antagonists administered during alcohol withdrawal can attenuate some alcohol effects. A) Administration of the NMDAR antagonist MK801 reduces the severity of alcoholrelated openfield hyperactivity (Thomas et al. 2001) but only when administered during the withdrawal phase (21 and 33 hours after alcohol exposure). Note that the activity levels, even of those treated with MK801 during the withdrawal period, do not reach those of the control subjects. B) Administration of the NMDAR antagonist eliprodil during the withdrawal period attenuated deficits on a spatial discrimination reversal learning task. Animals exposed to alcohol committed a significantly greater number of errors compared with all other groups. Animals exposed to alcohol and then treated with eliprodil during withdrawal committed significantly fewer errors compared with the alcohol group, reaching levels comparable with the controls ( Thomas et al. 2004).

Antagonism of Alcohol Effects on L1 Cell Adhesion Molecules
Alcohol also may interfere with normal neurodevelopment by reducing cell adhesion. Such celltocell contact is imperative for neuronal communica tion, influencing neuronal growth, development and survival, and, in particular, cellular migration and the development of axons and dendrites. Alcohol's ability to specifically inhibit the adhesive properties and axon and dendrite outgrowth mediated by the L1 cell adhesion molecule (CAM) has implicated L1CAM as a target for developmental alcohol neurotoxicity (Bearer 2001). Because of their capacity to disrupt ethanol's action on L1 adhesion, 1 octanol and other alcohols can pre vent ethanolinduced apoptotic cell death, growth retardation, and neural tube closure delays (Chen et al. 2001a) (see figure 3). These findings suggest that development of pharmacological agents that prevent alcoholinduced inhibition of L1 cell adhesion may serve as therapeutic agents to reduce the severity of FASD. The potential development of such agents recently has been enhanced by the identifica tion of the alcoholbinding site on the L1CAM (Arevalo et al. 2008).
In addition, peptide fragments derived from neurotrophic factors like glialderived activitydependent neuroprotective protein (ADNP) and activitydependent neurotrophic factor (ADNF) also can reduce alcohol's teratogenic effects, in part by antago nizing alcohol's inhibition of L1 cell adhesion (Wilkemeyer et al. 2002). The active peptide fragments, NAPVSIPQ (NAP) and SALLRSIPA (SAL), which are derived from ADNP and ADNF, respectively, have demon strated strong neuroprotection, pre venting alcoholrelated fetal death, physical alterations such as neural tube and ocular defects, reductions in brain weight and volume, neuronal cell loss including the loss of serotoner gic neurons, and even spatial learning deficits. Although NAP and SAL can protect against a variety of neural insults, the ability of NAP to protect against alcohol's teratogenesis is related to its ability to antagonize alcohol's effects on L1 cell adhesion. Peptide derivatives of NAP that lack neuro protective properties against other insults still can prevent alcohol ter atogenesis, whereas derivatives that weakly block alcohol's effects on L1 remain neuroprotective against other insults but are less effective in block ing alcohol teratogenesis (Wilkemeyer et al. 2003).

Neurotrophic Factors
Neurotrophic factors influence cell metabolism and growth, proliferation, differentiation, migration and matu ration of cells, and apoptotic cell death. Alcohol exposure during development may impair neurotrophic factor production. In addition, when immature neurons are exposed to alcohol, the newly developed cells may respond abnormally to certain guid ance or trophic factors (Lindsley et al. 2003). Alteration by alcohol to either of these mechanisms may lead to a pathological cascade of events, which may ultimately contribute to FASD.
In the presence of alcohol, the levels of these neurotrophic factors, includ ing insulinlike growth factor, nerve growth factor, basic fibroblast growth factor, brainderived neurotrophic factor, and glialderived neurotrophic factor, are reduced (e.g., Climent et al. 2002). Conversely, as noted with NAP and SAL above, the administra tion of many of these growth factors can ameliorate alcohol's teratogenic effects (e.g., Mitchell et al. 1999). Such effects can range from mitigating alcoholinduced motor deficits (McGough et al. 2009) to increasing rates of survival for neurons and their axons and dendrites (Barclay et al. 2005) (see table). Such a broad range of mitigative effects may well be attributed to the large overarching contributions of neurotrophic factors to the development of the CNS.
These findings highlight the poten tial of neurotrophic factors as possible treatments for FASD. It also is important to note that neurotrophic factors may reduce alcohol's teratogenic effects by promoting cell growth and survival, even if alcohol does not directly affect that neurotrophic system. One challenge with in vivo adminis tration of neurotrophic factors is protecting against alcohol's teratogenic effects without disrupting normal developmental events.

Antioxidants
Alcohol also may lead to cell death via an imbalance between the pro duction of damaging reactive oxygen species and the ability of cells to protect themselves with endogenous antioxidants (i.e., oxidative stress) (see figure 4). Developmental alcohol exposure induces extensive apoptotic cell death, which is preceded by an increase in reactive oxygen species and low levels of protective antioxi dants (Ramachandran et al. 2003). Successful protection against alcohol related growth retardation, physical anomalies, and neuropathologies has been illustrated with a number of antioxidant agents, including resveratrol from red wine, curcumin from tumeric, and epigallocatechin3gallate from green tea (Antonio and Druse 2008). Similarly, the superoxide dismutase/ catalase mimetic EUK134 (Chen et al. 2004

Nutritional Factors
As noted above, vitamins C and E possess antioxidant properties. Other nutrients also may influence alcohol's teratogenic effects. Outcomes among children exposed to alcohol during pregnancy vary widely, and some of this variation may be attributed to nutritional factors. For instance, higher rates of FASD are observed in countries where malnutrition is prevalent (May et al. 2000). It there fore is likely that malnutrition can worsen fetal alcohol effects. In fact, alcohol is known to interfere with nutritional supply to the unborn fetus.
In contrast, nutritional supplements may attenuate alcohol's adverse effects on fetal development. For example, zinc deficiency may contribute to some FASD. Although zinc supplementation in one study did not mitigate alcoholinduced cerebellar Purkinje cell loss following developmental alcohol exposure (Chen et al. 2001b), the administration of zinc has been shown to protect against postnatal mortality, fetal dysmorphol ogy, and cognitive impairments in the offspring of alcoholtreated dams (Summers et al. 2008(Summers et al. , 2009. Similarly, folic acid deficiency during pregnancy is well known to induce neural tube defects, and folate supplementation can help protect against these congen ital malformations. Prenatal folic acid supplementation mitigates many of alcohol's teratogenic effects, including growth retardation, physical anoma lies, and neuronal loss (Wang et al. 2009).
Administration of another nutrient, nicotinamide (one of the B complex vitamins), during or shortly after developmental alcohol exposure in mice, also protects against alcohol related apoptotic cell death, as well as contextual fear conditioning deficits and overactivity in the open field (Ieraci and Herrera 2006). Importantly, nicotinamide did not alter the phar macokinetics of alcohol. However, it should be noted that the doses of nicotinamide used in this study trans late to much higher doses than those used in clinical populations. Thus, it is not clear if nicotinamide's actions are functioning at a nutritional or pharmacological level. Nevertheless, the findings are intriguing. colleagues (2009, 2010) also have reported that prenatal choline supplementation can mitigate alcohol's teratogenic effects, decreasing the severity of alcoholrelated birth weight reductions, physical anoma lies, and alterations in behavioral development. Choline is an essential nutrient that may mitigate alcohol's teratogenic effects via various mecha nisms, acting as a methyl donor, as a precursor to components of the cell membranes, or as a precursor to the neurotransmitter acetylcholine.
Given the known damaging effects of nutritional deficiencies on the developing fetus, nutritional supple ments may serve as a relatively easy means to improve outcomes among alcoholexposed offspring. However, one must keep in mind that there are synergistic effects among nutrients. For example, iron deficiency can exacerbate prenatal alcohol effects on growth (Carter et al. 2007), and although iron supplementation could have mitigating effects on FASD, it also can impair zinc absorption. Therefore, a balanced multisupplement diet throughout pregnancy may be most effective. Finally, it is important to note that nutritional interventions may be effective even if alcohol is not inducing nutritional deficiencies; in other words, nutritional interventions may be effective whether or not they are compensating for a deficiency. Certainly, further investigations of nutritional interventions for reducing FASD are needed.

Treatments for Individuals With FASD
For the most part, the experimental treatments described above must be given during fetal development, during the alcohol insult, to be effective. For example, administration of antioxidants must occur during oxidative stress, and the administration of NMDA receptor antagonists is only effective when given during alcohol withdrawal. In a clinical setting, atrisk populations could be targeted for such therapeutic interventions. However, it is difficult to identify atrisk women and ensure treatment compliance. Prenatal treat ment(s) may therefore be difficult to implement. Instead, focusing inter ventions on alcoholaffected children may be much more clinically relevant. Indeed, some of the treatments that are effective during the alcohol insult still may be effective when administered after alcohol exposure. For example, some neurotrophic factors can enhance neuronal survival and synaptic con nections, and can improve behavioral outcomes even when administered after alcohol exposure (e.g., McGough et al. 2009).

Nutritional Supplementation
As mentioned above, administration of choline during prenatal alcohol exposure can reduce the severity of adverse physical and behavioral out comes. Choline also may be effective even when administered after the alcohol exposure has ceased and during periods of postnatal development. The first study to show the beneficial effects of choline following develop mental alcohol exposure administered choline for 3 weeks following birth in rats exposed to alcohol during gesta tion. Choline reduced the severity of alcoholrelated working memory deficits observed in adulthood, illus trating that choline's effects were long lasting (Thomas et al. 2000) (see figure  5). Postnatal choline supplementation (up to postnatal day 30) also can reduce the severity of overactivity in the open field, reversal learning deficits, spatial learning deficits, and impairments in trace classical condi tioning (i.e., learning conditioned responses to eye blinkeliciting stimuli and feareliciting stimuli) associated with developmental alcohol exposure (Thomas et al. 2004b;Wagner and Hunt 2006). More recently, Ryan and colleagues (2008) demonstrated that administration of choline from either postnatal days 1120 or 2130 could improve behavioral outcome. In fact, choline supplementation during adolescence/young adulthood in the rat (postnatal day 40-60) can improve cognitive performance. Notably, choline loading during this later developmental period did not improve simple spatial learning or normalize activity levels in the open field but did significantly reduce the severity of alcoholrelated working memory deficits. These find ings suggest that choline administra tion later in development may target the prefrontal cortex. Importantly, in all of these studies, behavioral testing occurred after choline treatment, so benefits were not related to acute effects of choline. These data hold promise that nutritional interventions may be effective among individuals with FASD. Researchers currently are investigating the changes in brain functioning following perinatal choline supple mentation and the mechanisms of choline's actions.

Pharmacological Interventions
CNS plasticity (the ability of the brain to change) continues through out the lifespan. Although CNS plas ticity can be compromised following developmental alcohol exposure, the use of agents that enhance plasticity still may be effective. For example, a developmental alcohol insult will impair the activation of cAMP response element-binding protein (CREB) (Krahe et al. 2009), which is necessary for memory formation and retrieval, and other components of neuronal plasticity. Administration of agents that inhibit the enzyme phosphodi esterase (PDE) can prolong CREB activation and facilitate longterm potentiation, a strengthening of synaptic connections that may con tribute to learning and memory. In addition, a study in developing ferrets found that PDE inhibitors adminis tered 1 week after an alcohol insult increased CREB activation and restored sensory cortical plasticity (Krahe et al. 2009;Medina et al. 2006). Thus, even though alcohol may impair baseline plasticity, it may be restored with appropriate interven tions. Targeting plasticity in such a manner may be particularly effective when measures to prevent drinking during pregnancy fail.

Environmental Interventions
Neuronal plasticity also can be enhanced with environmental enrich ment. A typical enriched environ ment will include social, motor, and 82 Alcohol Research & Health Figure 5 Choline supplementation during early postnatal development mitigates cognitive deficits induced by prenatal ethanol (i.e., alcohol) expo sure. Prenatal alcohol exposure significantly impaired performance on a working memory learning task among adult rats. When the alcohol exposed animals received choline during early postnatal development, well after alcohol exposure, this effect was mitigated. sensory stimulation using running wheels and toys, items that the animals can crawl into, play on, manipulate, and chew. Communal rearing, neonatal handling, and exercise also can act as enriched environmental factors. Stimulation using such enrichment programs is known to elicit a number of CNS responses, ranging from increases in neurotrophic factor levels to structural changes including dendritic arborization and neurogenesis to improved learning. A variety of environmental enrichment paradigms have been shown to improve behavioral outcomes, including motor, social, and cognitive functioning, fol lowing prenatal alcohol exposure (see Hannigan et al. 2007). For example, animals exposed to alcohol in utero and then reared in standard cages exhibit abnormal gaits. When alcohol exposed animals were reared in an enriched environment, their gait did not differ from that of controls (Hannigan et al. 1993). Similarly, exercise on running wheels can attenuate the adverse effects of developmental alcohol exposure on hippocampal plas ticity and learning (Christie et al. 2005). Certain brain regions also can be targeted using specific enriched envi ronment paradigms. For example, acrobatic motor learning can enhance functioning of the cerebellum and attenuate alcoholinduced motor skill impairments (Klintsova et al. 2000). Although animals exposed to alcohol during development initially were not as skilled as the controls, after days of training, they eventually were able to perform at control levels. Even though significant cerebellar Purkinje cell loss remained following early alcohol exposure, an increase in the number of synaptic connections for each of the remaining Purkinje cells was found, showing the capacity for synaptic plasticity (Klintsova et al. 2002). Such mitigative effects are persistent and can be observed weeks after environmental enrichment.
In contrast, some studies have shown that brains exposed to alcohol during development do not possess the same capacity for plasticity as non-alcoholexposed brains. For example, one study failed to find an increase in adult hippocampal neuro genesis following environmental enrichment in mice prenatally exposed to alcohol. In comparison, control animals reared in the enriched envi ronment showed a twofold increase in neurogenesis (Choi et al. 2005). Thus, it is important to determine how much plasticity is maintained by the alcohol exposed subject and what manipulations will be most effective in capitalizing on existing plasticity. Moreover, in addition to identifying experiences that can improve out come, one may need to identify and protect against adverse experiences, such as early stress, that may exacer bate alcohol's teratogenic effects (see Abel and Hannigan 1995). Clearly, the postnatal environment can be key in influencing the adverse effects of developmental alcohol exposure.

Conclusions
Individuals born with FASD suffer from a lifetime of physical, cognitive, and behavioral problems. Ideally, one would intervene at the time of alcohol exposure, directly preventing or reducing maternal alcohol consump tion. However, because prevention is not always possible, there is a need to seek effective treatments that will mitigate developmental alcoholrelated deficits. The insights gained to date from experimental models offer several candidates for the attenuation of deficits associated with prenatal alcohol exposure. Although translating these treatments to the clinical arena poses challenges, including the difficulty of administering treatments during prenatal periods as well as safety concerns, continued research and refinement may lead to viable clinical treatments. Moreover, some treatments, including nutritional, behavioral, and environmental manipulations, can be much more readily translated. Because some women around the world contin ue to drink alcohol during pregnancy, it is critical to continue pursuing effective interventions. The present findings suggest that various treatments have the potential to attenuate alcohol's People with HIV are now living longer and healthier lives. Still, challenges remain in preventing both infection with the virus and progression of the disease. This Alcohol Alert outlines the role that alcohol has in HIV/AIDS prevention, transmission, and disease progression and touches on recent efforts to reduce these strong, yet preventable, effects.